Una S. Ryan

Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence.

The cellular and subcellular sites of angiotensin converting enzyme (kininase II) in lung tissue and endothelial cells in culture were examined by immunocytochemical and immunofluorescence techniques. Converting enzyme is capable of inactivating bradykinin and of converting angiotensin I to its potent lower homolog, angiotensin II. Immunocytochemistry at the electron microscope level used goat anti- (pig lung and angiotensin converting enzyme) coupled to 11-MP (11-microperoxidase) via glutaraldehyde or to 8-MP (8-microperoxidase) via a bifunctional active ester, bis-succinyl succinate. The latter conjugate, which does not contain complex polymers, has been characterized in detail in terms of immunoreactivity and peroxidase activity.

Vaccine-Induced Antibodies Inhibit CETP Activity In Vivo and Reduce Aortic Lesions in a Rabbit Model of Atherosclerosis

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Isolation and culture of pulmonary artery endothelial cells.

It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood.

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.

Inhibition of neointimal hyperplasia by blocking αvβ3 integrin with a small peptide antagonist GpenGRGDSPCA

Abstract Purpose: Neointimal hyperplasia is a leading cause of restenosis after vascular procedures. Recent findings showed that smooth muscle cell (SMC) migration from the media into the neointima is a critical step in the development of the hemodynamically compromising neointimal lesion. Moreover, integrins are believed to play a role in SMC motility. Therefore we studied the role of one ubiquitous integrin, α v β 3 , in SMC migration. Methods: Transwell assay was used to study in vitro migration of human and rabbit SMCs after stimulation with platelet-derived growth factor (PDGF). A neutralizing monoclonal antibody to α v β 3 , LM609, and a specific arginine-glycine-aspartic acid (RGD) antagonist, G penGRGDSPC A, were used in the migration assay to inhibit α v β 3 -mediated SMC migration. In addition, G penGRGDSPC A was administered locally to rabbit carotidartery after balloon angioplasty to determine the effect of blocking α v β 3 on neointimal hyperplasia. Results: We showed that PDGF-induced human SMC migration is mediated by the α v β 3 integrin by use of LM609 to inhibit migration and that SMC migration is RGD dependent by use of G penGRGDSPC A to inhibit migration. We have also inhibited rabbit SMC migration with G penGRGDSPC A to demonstrate the cross-species preservation of the RGD peptide sequence in SMC mortality. Finally, when we administered G penGRGDSPC A locally to rabbit carotid artery after balloon angioplasty, there was a statistically significant reduction in neointimal lesion formation compared with arteries administered an inactive peptide or saline solution. Conclusions: We have demonstrated the important role of the α v β 3 integrin in SMC migration in vitro and in neointimal hyperplasia in vivo. (J VASC SURG 1994;19:125-34.)

Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes

Enzymes situated along the luminal surface of pulmonary endothelial cells interact with circulating solutes, notably with vasoactive substances, to regulate the hormonal composition of systemic arterial blood. However, it is becoming clear that the range and complexity of reactions occurring at or near the surface of endothelial cells are greater than previously recognized. In addition, evidence indicates that the quality of cell cultures used to define specific endothelial functions must be carefully controlled, together with development of improved understanding of the effects of long-term culture on pulmonary endothelial cells. We have developed new techniques for the culture of pulmonary endothelial cells which avoid exposure to proteolytic enzymes at both the isolation step and during subculture. A combination of mechanical harvest and culture on microcarrier beads has provided a system for the long-term, large-scale culture of pulmonary endothelial cells, features which to a large extent determine the scope of biochemical studies which can be undertaken.

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells.

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.

The safety and immunogenicity of a CETP vaccine in healthy adults

A cholesterol ester transfer protein (CETP) vaccine (CETi-1) that induces auto-antibodies that specifically bind and inhibit activity of endogenous CETP has been demonstrated in rabbits to significantly increase HDL-C and reduce the development of atherosclerosis. In a Phase I human trial with CETi-1, one patient at the highest dose (250 mg) out of a total of 36 patients who received a single injection developed anti-CETP antibodies. In an extension study of 23 patients, 53% (8/15) who received a second injection of the active vaccine developed anti-CETP antibodies compared with 0% (0/8) in the placebo group. The vaccine was well tolerated and no significant laboratory abnormalities occurred. CETi-1 is a feasible therapy in humans to induce CETP auto-antibodies. Future research will determine if repeat inoculations will induce a sufficient anti-CETP antibody response to inhibit CETP and increase HDL levels.

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

New Substrates for the Radioassay of Angiotensin Converting Enzyme of Endothelial Cells in Culture

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.